A guiding assumption in neurobiology has been that storage of information in the brain involves persistent, use-dependent alterations in neuronal electrical function. Determining the molecular basis of these forms of neuronal plasticity will provide the fundamental understanding necessary to provide therapies for diseases of memory as well as related persistent changes in electrical signaling that accompany addiction and epilepsy. Glutamate exerts its effects on the postsynaptic membrane through activation of two classes of receptor. Ionotropic glutamate receptors rapidly open integral cation channels while metabotropic receptors activate or inhibit G-protein coupled enzymes. Persistent modulation of fast neurotransmission, through both pre- and postsynaptic actions, comprises conventional LTP and LTD. Glutamate also acts upon postsynaptic mGluRs, particularly mGluR1 and mGluR5. Activation of mGluR1/5 stimulates phospholipase C2 and triggers a slow EPSC mediated by TrpC cation channels. Recently, we have reported that the mGluR1-mediated slow EPSC evoked by parallel fiber-Purkinje cell bursts shows strong LTD following either repeated climbing fiber-Purkinje cell synaptic activation or direct postsynaptic depolarization. Here, we propose to extend the characterization of this novel and unique form of synaptic plasticity. Aim 1. What molecular mechanisms underlie the induction of LTD(mGluR1) in Purkinje cells? We know that LTD(mGluR1) requires Ca influx for induction but we have a poor understanding of the parameters of the Ca signal. We shall use specific toxins of voltage- sensitive Ca channels to block LTD(mGluR1). This shall be done together with 2-photon Ca imaging of dendritic spines. We shall also attempt to induce LTD(mGluR1) by photolysis of postsynaptic caged Ca. We shall perform a screen to identify Ca-sensitive enzymatic processes (kinases, phosphatases, proteases, phospholipases) that may couple Ca influx to expression of LTD(mGluR1). Aim 2. What molecular mechanisms underlie the expression of LTD(mGluR1)? Is LTD(mGluR1) expression mediated by internalization of surface mGluR1? We shall test this by a) postsynaptically applying drugs and peptides to block mGluR1 endocytosis and b) transfecting Purkinje cells with our super-ecliptic-pHlorin-tagged mGluR1 to measure internalization with two-photon microscopy. The activity of mGluR1 is under dual regulation by Homer proteins and the prolyl isomerase PIN1. To test the hypothesis that this signaling cascade is involved in expression of LTD(mGluR1) we shall use a combination of drugs, peptides and knockout and mice. Aim 3. What are the consequences of LTD(mGluR1) for cerebellar circuit function &memory storage? We have shown that LTD(mGluR1) blocks the induction of conventional AMPA-R LTD at parallel fiber synapses. To extend analysis of meta-plastic effects, we shall assess the interaction of LTD(mGluR1) with short-term retrograde modulation of glutamate release that uses mGluR1-driven endocannabinoid signaling. PUBLIC HEALTH RELEVANCE: A guiding assumption in neurobiology has been that storage of information in the brain involves persistent, use-dependent alterations in neuronal electrical function. Determining the molecular basis of these forms of neuronal plasticity will provide the fundamental understanding necessary to provide therapies for diseases of memory as well as related persistent changes in electrical signaling that accompany addiction and epilepsy. Here, we propose to study the molecular mechanisms and functional role of a novel form of neuronal plasticity that we discovered recently, called "long-term synaptic depression of the neurotransmitter receptor mGluR1," abbreviated as LTD(mGluR1).